Progress in pathogenesis research of Ustilago maydis, and the metabolites involved along with their biosynthesis

Abstract Ustilago maydis is a pathogenic fungus that causes corn smut. Because of its easy cultivation and genetic transformation, U. maydis has become an important model organism for plant‐pathogenic basidiomycetes. U. maydis is able to infect maize by producing effectors and secreted proteins as well as surfactant‐like metabolites. In addition, the production of melanin and iron carriers is also associated with its pathogenicity. Here, advances in our understanding of the pathogenicity of U. maydis, the metabolites involved in the pathogenic process, and the biosynthesis of these metabolites, are reviewed and discussed. This summary will provide new insights into the pathogenicity of U. maydis and the functions of associated metabolites, as well as new clues for deciphering the biosynthesis of metabolites.


| INTRODUC TI ON
Ustilago maydis, a basidiomycete fungus, is a common biotrophic phytopathogenic fungus capable of infecting all aerial organs of corn plants.
It is parasitic exclusively on maize (Zea mays), one of the major cereal crops in the world, and its ancestor, teosinte (Z. mays subsp. mexicana).
This disease can hinder the growth of plants and reduce the yield, which leads to serious economic losses. The U. maydis-maize interaction process has effects at the cellular and molecular levels, leading to host cell physiology and metabolic disorders, manifesting as characteristic tumours formed by different host organs (Luo et al., 2020). The life cycle of U. maydis involves the germination of diploid teliospores on the host, followed by mating between haploid meiotic progeny to form invasive dikaryotic mycelia. The hyphae then form appressoria to invade host tissues, followed by extensive proliferation, tumour induction, and eventual formation of pigmented teliospores . To successfully colonize its host, U. maydis has developed various strategies to respond to the host, such as releasing effectors in response to the reprogramming of host metabolism (Cui et al., 2015;Djamei & Kahmann, 2012). To date, hundreds of effectors have been discovered, and the functions of some of the core effectors have been thoroughly characterized. The effectors act in multiple ways on different targets, such as suppressing plant immunity, manipulating plant physiology, and being recognized by host defence mechanisms, thus promoting pathogen infestation, expansion, and colonization. In the narrow sense, effectors are proteins secreted by pathogens into the extracellular and intracellular spaces of host plants (Duplessis et al., 2011;Gan et al., 2013;Giraldo et al., 2013;Saitoh et al., 2012).
In the broad sense, effectors are proteins and small molecules that alter the structure and function of host cells, thereby promoting the colonization of pathogens (Horbach et al., 2011). The rapid development of omics technologies has facilitated the discovery and functional characterization of an increasing number of effectors. The increasing deciphering of the U. maydis genome provides more valuable information for revealing the pathogenic mechanism (Han & Kahmann, 2019;Lanver et al., 2017;Redkar et al., 2017). Due to its highly divergent development and reverse genetic adaptations, U. maydis provides a model system for the genetics and cell biology of smut fungus, as well as for studies on the interaction between living trophic plant pathogens and plants (Zuo et al., 2019).
Although U. maydis is well known as a maize pathogen, its metabolites have many biological activities. Chemical composition investigation and activity identification studies have shown that U. maydis-derived ustilagic acids (UAs, 1-3 in Figure 1) (Kahmann & Kamper, 2004;Yang et al., 2013) and mannosyl erythritol lipids (MELs, 4-7 in Figure 1) contain uncommon glycolipids of cellobiose and erythrose with biosurfactant and antifungal activity . These compounds not only assist in the process of U. maydis appressoria adhesion to the plant surface, but also prevent damage to plants by other fungi (Kuiper et al., 2004). In addition, U. maydis was also found to yield other metabolites, such as ferrichromes (8-9 in Figure 1) (Neilands, 1981;Winterberg et al., 2010) and melanin (Reyes-Fernandez et al., 2021).
Herein, we summarize advances in the pathogenesis of U. maydis, as well as the metabolites and their biosynthesis associated with corn smut infection. This review provides a specific perspective on the pathogenicity of U. maydis that will contribute to a comprehensive understanding of corn smut.

| THE LIFE C YCLE OF U. mayd is AND ITS PATH O G E N I CIT Y
Many fungal plant pathogens cause drastic morphological changes in host organs due to host cell proliferation and overgrowth (Wildermuth, 2010). The smut fungi Ustilaginales have been found to cause extreme changes in host tissue morphology (Luttrell, 1981).
U. maydis has become a model microorganism used to study the mechanisms of interactions between biotrophic fungi and plants (Bölker, 2001). Considered as one of the top 10 plant fungal pathogens, U. maydis can infect all aboveground organs of the maize plant, including seedlings, ears, and adult leaves, and induce tumour (commonly known as maize black truffles) formation and become a significant threat to modern maize productivity (Dean et al., 2012). The parasitism of U. maydis does not lead to the death of maize plants ( Figure 2a). However, when the infection is severe, maize does not spit ears (Figure 2b), and further forms massive tumour tissue ( Figure 2c). To successfully colonize hosts, U. maydis has developed several strategies including evasion of host recognition, interference with plant defence responses, and reprogramming of host metabolism (Redkar et al., 2017).
The two-stage life cycle (Figure 2d) of U. maydis is closely related to its infection process, which has been revealed by numerous investigations. First, haploid spores undergo saprophytic growth and germinate on specific substrates to produce yeastlike colonies. However, this form is not pathogenic (Figure 2d, I).
Then, the compatible haploid cells form a conjugation tube ( Figure 2d, II) and fuse to form an invasive binucleate mycelium (Figure 2d, III) (Kahmann & Schirawski, 2007). In the early stage of infection, the ends of the binucleate hyphae of U. maydis, known as appressoria, swell and begin to penetrate the epidermal cells of growing maize (Figure 2d, IV) (Lanver et al., 2014;Snetselaar & Mims, 1993). These filaments differentiate into infection structures known as appressoria. After penetration of the epidermal layer of the plant, the cell cycle arrest ceases and clamp-like structures ensure the correct separation of two different nuclei and maintain the dikaryotic state in the growing hyphae (Lanver et al., 2017). The extracellular mycelium grows between cells without causing visible disease, while the intracellular mycelium is tightly surrounded by the plant plasma membrane, forming a living nutrient interface that facilitates the exchange of nutrients and signalling molecules, including various proteins (Matei & Doehlemann, 2016) (Figure 2d, V). This process involves the secretion of hundreds of effectors by U. maydis in various ways to suppress the plant's innate immune system and manipulate host metabolism (Cui et al., 2015;Djamei & Kahmann, 2012;Han & Kahmann, 2019). Subsequently, the mycelium proliferates massively in the plant foliar tissue, vascular system, and surrounding cavities, resulting in the formation of plant tumours (Figure 2d, VI). Next, extracellular hyphae form large aggregates in the cavities between plant tumour cells, the nuclei of binucleate mycelium cells fuse, and the mass proliferating hyphae break off and form pigmented aggregates of spores (Matei & Doehlemann, 2016). When the tumours dry up and rupture, the spores are released (Figure 2d, VII) and germinate under suitable conditions. The nuclei of diploids undergo meiosis and germination to form promycelium and haploid spores. The entire life cycle of U. maydis is strictly dependent on the plant, and usually takes about 2 weeks (Lanver et al., 2018).

| EFFEC TOR S INVOLVED IN PATHOG ENI C PRO CE SS E S
Maize undergoes a series of physiological and biochemical changes in response to U. maydis infection (Wang et al., 2007). Doehlemann et al. investigated maize's defensive response to U. maydis infection using transcriptome and metabolome analysis, revealing that secondary metabolite production was activated, cell division mechanisms were increased, and pathogenesis-related genes were induced (Doehlemann, Wahl, Horst, et al., 2008). Zou et al. analysed transcriptomic data on the early response to U. maydis infection of maize, revealing specific changes in maize cells during tumour formation (Zou et al., 2022). In brief, when infected with U. maydis, maize strengthens its defensive mechanism through physiological changes (Djamei et al., 2011;Li et al., 2016). However, during an infection, U. maydis suppresses the plant's defensive response by secreting effectors (Win et al., 2012). Effectors released by pathogens are secreted proteins that affect the structure and function of host cells, thus facilitating the success of the pathogen infestation process.

| The type of function of the effectors
Most effectors are secreted proteins generated by pathogenic strains, and genome sequencing has tremendously aided in effector discovery and identification. The effectors have different functions due to different locations, and apoplastic effectors play a role in the interaction area between fungal hyphae and the host (Stotz et al., 2014). Generally, the targets of these apoplastic effectors exist in the first layer of the plant defence response. Most apoplastic effectors are plant cell wall-degrading enzymes (PCWDEs), which can destroy the structure of host cell walls and facilitate hyphae invasion (Chen et al., 2021;Quoc & Chau, 2017). The effectors located in the cytoplasm can regulate host resistance by regulating the maturation and localization of plant resistance proteins (Qi et al., 2019). In addition, interfering nucleoprotein can directly regulate the transcription of many immune-related genes. Some nuclear effectors have been well characterized in plant-pathogenic fungi (Wu et al., 2022).
The decryption of U. maydis 521 genomic information revealed that 18.6% of the genes encoding secreted proteins are distributed in 12 clusters of the genome, ranging from three to 26 genes per cluster. These genes were up-regulated in expression on U. maydis infestation of plants . U. maydis can produce 467 putative secreted effector proteins, which can promote the colonization of U. maydis to the host (Lanver et al., 2018). The finding of these secreted proteins has established the groundwork for further research into U. maydis effectors. Furthermore, effectors respond to particular processes happening in the host, such as host reprogramming, to increase infection Lanver et al., 2017). Because U. maydis lacks specialized feeding structures, signal exchange and nutrient absorption must occur via biotrophic interfaces (Brefort et al., 2009). Despite substantial transcriptome research revealing the spatiotemporal-specific expression of U. maydis effector genes (Lanver et al., 2018;Mine et al., 2018), only a few critical effectors have been functionally characterized (Table 1) Hemetsberger et al., 2012). Hemetsberger et al. (2015) categorized Pep1 as a phylogenetically conserved core effector of U. maydis, noting that this effector may play an essential role in biotrophic smut fungus pathogenicity.
Rip1 is an effector whose function has not been fully elucidated.
It inhibits the function of pathogen-associated molecular pattern (PAMP)-triggered ROS, thereby reducing host immunity . Late effector protein 1 (Lep1) is a novel core effector that is highly expressed during tumour formation (Fukada et al., 2021).
The core effector Rsp3 is a critical virulence factor that protects mycelium from maize DUF26-domain family proteins (AFP) proteins (Ma et al., 2018). Another core effector, Jsi1, interacts with the plant corepressor family Topless/Topless related (TPL/TPRs) proteins by increasing the transcription of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signalling pathway, thereby promoting virulence and suppressing the host immune system (Darino et al., 2021). Cce1 is a recently identified core effector that is localized in the plasmodial ectodomain; however, its target has yet to be identified (Seitner et al., 2018). Naked1 (Nkd1) is an effector localized in the nucleus that was identified by Navarrete et al. (2022). By binding to transcriptional co-repressors TPL/TPRs, Nkd1 not only blocks auxin signalling and recruitment of transcriptional repressors but also leads to activation of auxin and JA signalling and promotes susceptibility to (semi-)biotrophic TA B L E 1 Key effectors of Ustilago maydis and their function. pathogens . Hoang et al. (2021) found that virulence promoting 1 (Vp1) is another novel secreted effector protein. Vp1 is localized to the plasmodial ectodomain and promotes its own virulence and ability to colonize the host (Hoang et al., 2021).
U. maydis can also secrete other effectors to ensure its successful colonization on the host. The translocation effector See1 is transferred to the maize cytoplasm and nucleus by the mycelium. In the cytoplasm, it interacts with SGT1 in maize and interferes with mitogen-activated protein kinase (MAPK)-induced phosphorylation of SGT1. In the nucleus, See1 is required for the reactivation of DNA synthesis. Finally, it promotes the formation of tumours in seedling leaves . The effector Pit2 inhibits the activity of the key virulence target maize cysteine protease (CP1A, CP1B, CP2, and XCP2), which is required for the formation of tumours, while mutant pit2 strains induce a strong defence response in the host, suppressing mycelial spread and tumour formation (Doehlemann et al., 2011;Mueller et al., 2013).
Cmu1 is a secreted effector (Djamei et al., 2011) that can be trans- Effectors are involved in almost all processes of corn smut infection of plants. They are constantly under selection pressure from the host immune system. As a result, genes encoding effectors are the fastest evolving genes in pathogen genomes (Stergiopoulos et al., 2007). Because of the lack of identifiable structural domains and functional redundancy among effectors, functional identification of effectors is difficult. As a consequence, the functions of a large number of effectors (Table S1)

| Synthetic regulation of effectors
When the yeast-like haploid spores are transformed into dikaryotic hyphae, U. maydis is able to parasitize living plants. This dimorphic transformation is closely related to the process of infecting host plants, so effector expression regulation is essential for hyphal development (Lanver et al., 2017). The A and B alleles of the quadruple mating type system control this transformation process, with the A site controlling haploid spore mating and the B site determining the pathogenicity of the dikaryotic hyphae. Following the formation of the dikaryotic hyphae, the appressoria form in the proper position and begin the colonization process. Following that, the bE/bW heterodimer regulates the hyphal formation and pathogenic development (Brachmann et al., 2001;Müller et al., 2003).
Pheromone responses, bE/bW-mediated transcriptional regulatory networks, and surface signal sensing pathways all influence U. maydis effector expression . There have been numerous research reports on the regulation and regulatory network of effector synthesis (Brefort et al., 2009;Lanver et al., 2017). The key regulatory genes that are helpful to the U. maydis infection process are shown in Table 2.
Pheromone receptors Pra1 and Pra2 on the surface of host cells can sense pheromones and transmit signals to the cAMP and MAPK signalling pathways . These two signalling pathways further transmit the signal to the transcription factor Prf1, which causes the b site to be expressed, resulting in the formation of a bE/bW heterodimer (Kaffarnik et al., 2003). The bE/bW heterodimer can regulate the downstream transcription factor network through the transcription factor Rbf1 (Heimel, Scherer, Vranes, et al., 2010).
Early in the infection process, gene regulation of the zinc finger transcription factor Rbf1 is known to be required for the initial steps ing of host defences, such as kpp6 , cluster 5B , and pep1 (Hemetsberger et al., 2012;Molina & Kahmann, 2007).
In the late stage of infection, the main functions of effectors are to regulate processes such as tumour maturation and black teliospore production. There are few studies on the transcription factors regulating these late events. Ros1 down-regulated several critical effectors involved in the early colonization process and up-regulated the late expression of 51 novel effectors (Tollot et al., 2016). The transcription factor Nlt1 is also an important regulator, but only in lobe tumours (Lanver et al., 2018). At this stage, some regulatory genes or gene clusters can regulate the infection of U. maydis, such as clp1 , cluster 19A , and str1 ) that can make hyphae proliferate and branch in the host; and gpa3 (Krüger et al., 2000), ubc1 (Gold et al., 1997), rtf1 (Banuett, 1991), ust1 (García-Pedrajas et al., 2010) and ssp1 (Huber et al., 2002) that make the host form tumour-like tissue and produce spores.
The secretion of effectors during the infection process brings extensive pressure to the endoplasmic reticulum. After the effectors are regulated by transcription factors, they undergo protein modification in the endoplasmic reticulum and the Golgi apparatus.
The unfolded protein response (UPR) is a conserved eukaryotic signalling pathway that detects misfolded proteins in the endoplasmic reticulum. There is significant crosstalk between the UPR pathway and the bE/bW regulatory cascade (Lanver et al., 2018;Tollot et al., 2016). After U. maydis hyphae penetrate the plant epidermis, the UPR is activated immediately and participates in the regulation of the next step of gene regulation . After modification, effectors are secreted into the apoplast and host cells to function. For example, U. maydis secretes the effector Cce1 to attenuate callose deposition in the cell wall (Matei & Doehlemann, 2016), and the metalloprotease Fly1 can destroy chitinases in host plants (Ökmen et al., 2018). The conserved effector protein Erc1 binds to host cell wall components and exhibits 1,3β-glucanase activity, which attenuates the β-glucan necessary for the induced defence response (Ökmen et al., 2022).

| Effector and omics research
U. maydis has become one of the most dangerous diseases in maize production due to the rampant occurrence of smut diseases and the decreasing resistance of crop varieties. Genome sequencing of U. maydis is indispensable for a comprehensive understanding of the TA B L E 2 Key regulatory genes in the pathogenic process of Ustilago maydis.  U. maydis genomes have been sequenced and published (Table S2), which not only provides valuable clues for in-depth interpretation of the genome of U. maydis, but also provides an in-depth understanding of the secondary metabolites.
Recent developments in transcriptomics, as well as comparative genomics, have facilitated the in-depth study of effectors, and many studies are no longer limited to the functional analysis of individual effectors, but rather to the comparative and in-depth analysis of the functions of proximate effectors and their evolutionary history .
The discovery of the effectors' functions contributes significantly to the understanding of the corn smut infection mechanism. Secondary metabolites produced by U. maydis, in addition to effectors, increase its pathogenicity. MELs and UAs are presumed to help appressoria adhere to the smooth hydrophobic surfaces of host plants (Haskins, 1950;Kuiper et al., 2004;Lemieux & Charanduk, 1951). Thus, secretion of metabolites like MELs and UAs are thought to play important supporting roles in the processes of hyphal attachment and infestation of plant surfaces (Feldbrügge et al., 2013). Additionally, siderophore (Mei et al., 1993) and melanin (Islamovic et al., 2015) were found to be involved in the infection processes of U. maydis.

| S ECONDARY ME TABOLITE S INVOLVED IN PATHOG ENIC PRO CE SS E S AND THEIR B IOSYNTHE S IS
The production of secondary metabolites by fungi is not without merit. For example, the polyene compounds secreted by mushrooms can inhibit insect bites and are a class of efficient chemical defence molecules (Brandt et al., 2017). U. maydis also produces some specific secondary metabolites to facilitate its infection of maize. To date, 25 secondary metabolites have been reported, including UAs (1-3), MELs (4-7), ferrichromes (8-9), melanin precursor (11-16), and other compounds (17-25) (Figure 1), some of which are associated with the pathogenesis of corn smut.

Ustilagic acid C (3) was isolated as a new component from U. may-
dis treated with chemical epigenetic modifiers (Yang et al., 2013).
UAs were the first glycolipids to be discovered with cellobiose, a rare  (Figure 3a) (Teichmann et al., 2007(Teichmann et al., , 2010. UA production after supplementation with 16-hydroxypalmitate, thus indicating that Cyp1 is responsible for the terminal hydroxylation of palmitate. A cyp2 mutant produced UA lacking hydroxyl groups, indicating that Cyp2 is responsible for the minor terminal hydroxylation of palmitate. The ahd1 mutant secretes UA lacking α-hydroxyl groups, indicating that Ahd1 is responsible for the α-hydroxylation of UA (Teichmann et al., 2007). Knockout of cyp2 revealed that the UA produced by the cyp2 mutant strain lacked the hydroxyl group, suggesting that Cyp2 is responsible for the secondary terminal hydroxylation of palmitic acid. When rua1 is knocked out, UA is not produced. The biosynthetic pathway of UAs is summarized in Figure 3b.

| MELs and their biosynthesis
MELs are well-known biosurfactants and were discovered in the metabolites of Ustilago sp. as early as 1955 (Haskins et al., 1955).
Structurally, the backbone of MELs is a disaccharide composed of mannosyl and erythritol, and C4 and C6 of the mannosyl moiety are linked to short-chain and medium-chain fatty acids through ester groups (Deinzer et al., 2019). MELs are frequently classified as A, B, C, and D (4-7 in Figure 1) due to differences in the number of acetyl groups attached to C4 and C6, as well as their appearance order on thin-layer chromatography. MEL-A (4) represents a diacetylated compound (over 70% of MELs), while MEL-B and MEL-C are monoacetylated at C6 and C4, respectively, and the fully deacetylated structure is called MEL-D (Rau et al., 2005). Hewald et al. (2006) used the homologous recombination method (Kamper, 2004) for U. maydis genetic manipulation previously developed to knock out the acyltransferase-encoding genes mac1 and mac2, and the acetyltransferase-encoding gene mat1 on the putative BGC (Figure 3d). Among the mutants obtained, the Δmat1 strain selectively produced MEL-D, while neither the Δmac1 nor the Δmac2 strains produced any MELs. Therefore, two acyltransferases are considered to be necessary for the production of MELs. (Hewald et al., 2006). In addition, this predicted BGC also contains a glycosyltransferase Emt1 and a membrane transporter Mmf1 The biosynthetic gene clusters of MELs have been investigated in U. maydis in addition to being analysed, predicted, and identified in Ustilago hordei (Deinzer et al., 2019), Pseudozyma antarctica (Saika et al., 2018), and Aspergillus nidulans (Hewald et al., 2006).
The BGCs of MELs from these various species show high homology (Figure 3d).

| Siderophores and nonribosomal peptides
Most microorganisms produce a group of low-molecular-weight compounds called siderophores during iron stress, which have a particular affinity for iron acquisition and storage (Neilands, 1981). An evaluation of the role of siderophores on U. maydis in its pathogenicity showed that a high-affinity iron uptake system involving siderophores is indispensable (Mei et al., 1993). Ferrochrome (8 in Figure 1) and ferrochrome A (9) are two siderophore derivatives produced by U. maydis (Haas et al., 2008;Yuan et al., 2001) that are cyclic hexapeptides consisting of three δ-N-acyl-N-hydroxy-ornithine and three amino acids, glycine, serine, and alanine (Winkelmann, 2007;Yuan et al., 2001). The modification of the side chain of the ornithine residue and the difference in the species of the other three amino acids that form the cyclic peptide results in different siderophores (Bushley et al., 2008;Haas et al., 2008;Winkelmann, 2007).
Transcriptome analysis based on whole-genome microarrays identified three gene clusters related to the high-affinity iron uptake system and determined that the iron permease Fer2 plays a vital role in this system. Strains with normal fer2 function have a selective growth advantage under iron (III)-limiting conditions compared to strains with inactive fer2, thus suggesting that a high-affinity iron uptake system based on permeases plays a key role in the virulence of U. maydis (Eichhorn et al., 2006). Ornithine monooxygenase Sid1 and non-ribosomal peptide synthase Sid2 are the first identified functional genes for ferrichrome biosynthesis in U. maydis. Sid1 catalyses the first step in siderophore biosynthesis, and the inactivation of sid1 results in a lack of ferrichrome production (Mei et al., 1993;Wang et al., 1989;Yuan et al., 2001). Subsequently, four new members involved in the biosynthesis of ferrichrome A in U. maydis, fer3, fer4, fer5, and hcs1, were discovered. These genes constitute the bulk of the BGC. This BGC encodes six functional proteins, a nonribosomal peptide synthase Fer3, an enoyl-CoA hydratase Fer4, a hydroxyornithine acylase Fer5, two transporters Fer6 and Fer7, and a hypothetical gene, fer8 (Figure 4a) (Winterberg et al., 2010). Highperformance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the metabolites of individual mutants of fer3, fer4, and fer5 found that their inactivation all resulted in the absence of ferrichrome A, which suggested that these three genes are required for ferrichrome A production. Overexpression of the HMG-CoA synthase Hcs1 in U. maydis leads to an increase in ferrichrome A production. In-depth analysis revealed that Hsc1 associates with Fer4, implying that the product of Hsc1 is a substrate for Fer4 (Winterberg et al., 2010). ornithine catalysed by Fer5. Finally, methylgutaconyi hydroxy ornithine is cyclized with specific amino acids by Fer3 to form ferrichromes ( Figure 4b) (Winterberg et al., 2010).

| Melanin and polyketides
The formation of spores in plant tumour-like tissues was found to be associated with melanin (Islamovic et al., 2015). The production of melanin in U. maydis is catalysed by the laccase Lac1 and the polyketide synthase PKS1 during tumour formation (Islamovic et al., 2015).
Additional PKSs also contribute to melanin formation, which is present on chromosome 12 and expressed during late stages of infection (Lanver et al., 2018). Melanins polymerized as DHN or L-DOPA units are common melanins in fungi, with the former being more prevalent (Tsai et al., 1999;Woo et al., 2010). The discovery of two PKSs in U. maydis has been hypothesized to be associated with the melanization of teliospores (Islamovic et al., 2015).  Reineke et al. (2008) proved that U. maydis could efficiently produce indole-3-acetic acid (IAA, 17 in Figure 1) from tryptophan (Trp), thus confirming the prediction that U. maydis could synthesize IAA (Moulton, 1942). Although the synthesis of IAA by U. maydis is enhanced when maize plants are infected, the evidence that the IAA produced by U. maydis was involved in tumour formation was still minimal: U. maydis could still induce tumour formation despite the simultaneous knockout of four essential genes, tam1, tam2, iad1, and iad2, required for IAA biosynthesis (Doehlemann, Wahl, Vranes, et al., 2008;Reineke et al., 2008). In addition, U. maydis was found to efficiently convert exogenously added Trp to IAA (Basse et al., 1996).

| Miscellaneous compounds
Plant hormones produced or modified by microorganisms have been shown to play a role in plant disease occurrence. U. maydis produces two phytohormones, abscisic acid (ABA, 18) and cytokinins (CKs) (Morrison et al., 2015), with the former playing a role in the growth of U. maydis (Mills & Staden, 1978;Moulton, 1942). No fewer than 10 CKs have been identified from U. maydis (Morrison et al., 2015); a representative CK (19) is shown in Figure 1. A survey of CK and ABA levels during U. maydis pathogenesis clearly shows that CKs and ABA can accumulate in U. maydis-infected maize tissues and that levels are higher in tumours formed by U. maydis than in other maize tissues (Bruce et al., 2010).
Indole pigments such as pityriacitrin (20) (25) is involved in infection remains unknown, despite the literature claiming that organic acids contribute to smut infection (Kretschmer et al., 2022).

| D ISCUSS I ON AND PER S PEC TIVE S
The dimorphic life cycle of crop smut is naturally related to the process of infecting maize, starting from the adhesion of appressoria to the maize surface to the formation of new spores in tumour tissue. This part of the cycle of parasitizing maize plants demonstrates the mycelial shape. The interaction process between the myceliumlike smut and maize has been profoundly studied at the cellular and molecular levels. The effectors secreted by U. maydis inhibit the rearrangement activity of maize due to infection and facilitates its infection. U. maydis additionally assists the adhesion of appressoria to the surface of plants by producing lipopolysaccharide compounds with surfactant properties. Genome sequencing of multiple U. maydis isolates will further facilitate the discovery of more effectors.
It will also provide favourable conditions for the biosynthesis of pathogenicity-related secondary metabolites.
Structurally diverse secondary metabolites of U. maydis, including UAs, MELs, ferrichromes, and melanin, have been found to be extensively involved in the pathogenicity process of U. maydis. These compounds have also been shown to have antibacterial and antitumour properties, antioxidant activities, and biosurfactant and other biological functions. It is often considered that secondary metabolites are not of much significance to their producers. However, this is not absolute, as in the case of U. maydis and its secondary metabolites.
It has been shown that as far as the structural type of secondary metabolites is concerned, U. maydis as a basidiomycete differs significantly from that of mushroom-form basidiomycete species like Laetiporus spp. (Duan et al., 2022) and Inonotus hispidus , and the number of secondary metabolites is much less.
Although the genome of U. maydis is predicted to contain multiple BGCs for secondary metabolites ( Figure S1), not many compounds have been isolated from U. maydis. This implies that most BGCs of U. maydis are inactive, which is also supported by the silent BGCs of some metabolites in U. maydis found in the few biosynthetic studies.
In conclusion, this work summarizes the progress of pathogenicity studies, effectors and their assistance in pathogenesis, as well as secondary metabolites involved in the pathogenic process and their biosynthesis by U. maydis. It summarizes the pathogenesis and players of crop smut from a unique perspective, and provides systematic insights for understanding the pathogenicity.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed.